Studies On, the Nature of the Growth Stimulus in Cancer

STUDIES ON, THE NATURE OF THE GROWTH STIMULUS IN CANCER

MONTROSE T. BURROWS

(From the Research Laboratories of the Barnard Free Skin and Cancer Hospital, and the Department of Surgery, Washington University School of Medicine, Saint Louis, Missouri) Our general studies of growth as seen by the tissue culture and an analysis of the conditions which induce and cause the growth of the cells in cancer have indicated quite clearly that cancer is not a reversion of the cells to an embryonic type, but evidently the freeing of them from those general forces which hold them together to make the organism a unit structure or, in other words, a return of them to their most primitive unicellular existence. Cancer does not deal with questions of embryology, but those much more general evolutionary forces which came into existence and led to the development of metazoan forms of plants and animals. The facts which have forced this generalization were derived by finding certain very definite similarities between cancerous tissue and cultures of unicellular organisms which show these tissues and cells to be living under very different conditions than the cells in the normal organism. These facts are the outcome of an analysis of the processes of growth, differentiation and function of body cells, as they exist in the tissue culture, in the normal organism and cancer. Many of the earlier students of botany and general biology had fully appreciated that the cell is not the dominating agent in the development of the body. Its growth, differentiation and function as they occur in the body are initiated and regulated by other much more general formative forces in the organism. This idea is found in the works of the botanists, Hoffmeister, Sachs and DeBary and also in the studies of Driesch, Whitman, Hertwig and other students of animal life and development (1). 239 240 MONTROSE T. BURROWS Ribbert had also appreciated this general fact in his studies of cancer. Others in biology and medicine have taken a different view. They have looked at the cell as the dominating agent. The cycle of change peculiar to the cell in the body is controlled by it. Those clinging to the first view looked at cancer as merely the freeing of the cell from the dominance of these general forces or the result of changes in the environment about the cells. The others looked at the cancer cell as a different cell type. Before the solution of the problem of cancer could proceed it was necessary that this controversy be settled. It remained for the tissue culture to give the facts necessary for the decision. As noted in 1915 (2) the author had found that, whether an embryonic heart muscle cell is to grow, to differentiate or to function depends entirely on its immediate environment in the tissue culture. The heart muscle cell can be changed from one state of activity into the other at will, by changing the environment about it. It was also interesting to note that these cells when they grow in a tissue culture do not grow to form organs. Their growth is not followed by differentiation. They grow as cancer cells grow in the organism and as bacteria grow to invade the medium and degenerate in cultures and in nature. It became evident, therefore, from these early observations that the growth of the cells in the body, their differentiation and function is something guided absolutely by other general forces in the organism and that these cells in their simplicity are not fundamentally different from other simple unicellular organisms. When isolated under conditions suitable for their growth they grow as these simple unicellular organisms grow. Cancer in the light of these facts cannot be a reversion of the cell to an embryonal type, but rather a return of the cell to its more primitive state or a release of it from those forces dominating it in the organism. The questions that these studies then presented were the nature of the dominating forces in the organism and the conditions which freed the cells from them and allowed them to grow independently as in cancer. The solution lay in the NATURE OF THE GROWTH STIMULUS IN CANCER 241 more complete analysis of the conditions in the environment controlling each of these changes, growth, differentiation and function. In a previous paper (3) the author had already shown that the cells of young embryos from 60 hours to 10 or 11 days old grow most readily in tissue culture. In studying the growth of cells of these embryos it was shown that it takes place in normal blood plasma and simple synthetic medium only when these cells are crowded into narrow stagnant confines which are supplied with an ample amount of oxygen. This growth can be slowed or stopped by washing these cells with a stream of serum or salt solution. It can be stopped by separating the cells or increasing relatively the amount of plasma or other media about them without disturbing their oxygen supply. When too few mesenchyme cells are placed in a drop of plasma for growth to take place, they migrate apart and show changes peculiar to their differentiation in the body. As these cells separate more and more their movements slow. Finally, they come to rest and then lie inactive for an indefinite time. These inactive cells are physiologically dead, but fully intact. When collected together again and placed in fresh plasma, they show activity as before. Single mesenchyme cells placed in a layer of plasma, may stretch out to a stellate or spindle shape and then come to rest. For these cells to show any great activity they must be crowded with other cells. Function maintains in these body cells when one end of the cell is placed against a dense stagnant mass of cells and the other end is in contact with the open medium into which any products liberated by the cells in its metabolism may escape. This fact was deduced by the study of heart muscle cells of chick-embryos in the tissue culture. When these cells are crowded together, they revert to simple growing mesenchyme cells. When they are less crowded they differentiate into fibroblasts. When one end is placed against a dense mass of cells or is bathed with products diffusing from such a dense mass of cells and the other end is in contact with the open medium into which any products of the metabolism of these 16 242 MONTROSE T. BURROWS cells may escape they contract rhythmically. .Any type of muscle cells may be made at will, to contract rhythmically, to change into a simple mesenchyme-like cell capable of forming intercellular fibrils or to grow and divide. These particular environmental conditions are important not only for the growth, differentiation and function of muscle cells, but for all types of cells in the body. The type of function is not determined by the environment, but by the cell. Isolated heart muscle and striated muscle cells contract at a rate of 90 per minute. Their contractions are quick and snappy. Single smooth muscle cells on the other hand have the same slow contractions peculiar to them in the body. In the body all functioning cells have a similar polarity. The nerve fibrils are stretched between the dense brain tissue and an end organ. The gland cells are stretched between a stagnant duct and a rich vascular supply without. From these observations it became evident, therefore, that the conditions suitable for the growth of body cells are not different from those of unicellular organisms. It is in the stagnant pool and not in the running stream that such life abounds. Wilders had shown that a few yeast cells will not grow in a wine cast. Either many or an extract of yeast must be added before growth takes place. The same has been shown to be true for many bacteria. Differentiation and function as noted above in the case of body cells are changes which take place under conditions not suitable for growth. They are the result of less crowding on the one hand and a localization of stagnant conditions on the other. In analyzing for the significance of these facts it has been found that stagnation and cell crowding are important for the growth of these cells because this growth depends on the accumulation about the cells of a certain concentration of a substance or substances formed by them. This substance or substances has been called the archuaia (8). It cannot be retained by the cells. It is soluble in the circulating fluids of the body, in salt solution, in serum and in plasma. It can NATURE OF THE GROWTH STIMULUS IN CANCER 243 reach the concentration necessary for growth only when there are a sufficient number of cells forming it in a small quantity of medium from which it cannot escape. This substance can be readily extracted from growing systems and added to medium containing isolated and inactive cells. From these studies it was learned that it acts not only to produce growth, but every act of the cell. When a fragment of cellular tissue is placed in a culture medium the cells first show migration, then growth, and finally self digestion as the archusia gradually accumulates about them. A study of the effect of the archusia on isolated cells has shown that it has no effect in low concentrations (Sl). In medium concentrations (S2)it causes the cells to migrate into plasma clots, to engorge themselves with fat droplets and protein particles. Extracellular fibrils of connective tissue are laid down under these conditions. Function takes place when this concentration of the archusia is maintained at one end of the cell. Growth and division never maintained under these conditions. It is in concentration (S3) that the cell digests the fat and protein in its cytoplasm and the medium about it, grows and divides. In all higher concentrations (R4) it suffers a self-digestion. This self-digestion although similar morphologically to autolysis is not the same. Toxic products are liberated in the autolysis of cells from the absence of oxygen. No such toxic products are liberated in this self digestion of the cell. It is similar to the degeneration seen in the center of malignant tumors. It was in the light of these facts that the author assumed that cancer may be nothing more than the result of a local crowding of cells in the organism and a relative reduction in the blood supply to the mass thus formed. In proof for such a contention it was found that those conditions which lead to cancer act only in this capacity. Recent careful work on the cause of cancer has shown very definitely that it may be produced by any one of a number of conditions and substances; such as coal tar, X-ray, radium, animal parasites, bacteria, etc. In man it arises most frequently on old inflammatory processes and in congenital tumors and defects. 244 MONTROSE T. BURROWS There was no evidence from these same studies that any one of these substances act otherwise than to induce this disease. Coal tar applied repeatedly to the skin produces cancer. Cancers thus produced then proceed independently of the tar. The same is true for cancers produced by animal parasites, X-rays and radium. These studies alone have shown that cancer is not the result of any specific external agent, but merely the result of some change in the tissues or the cell which may be produced by any one of a number of different substances and conditions. In previous studies (4) the author had shown that body cells have no mechanism for migration such as is seen in the cilia of paramoecium or many bacteria or in the internal mechanism of the amoeba. They migrate by liberating a substance which is readily absorbed by proteins and fats. This decreases the surface tension of the cells in the presence of proteins and fats. Body cells cannot migrate into liquids, but only into masses of protein and towards large fat droplets. Smaller particles of protein and small fat droplets are drawn into the cell by the same mechanism. This substance is a lipoid and has been called the ergusia (L). It is soluble in coal tar and many other lipoid solvents. Jorstad (5) has found that drops of coal tar placed in the tissue dissolve this substance and drag the tissue cells to them away from their intercellular substance and blood vessels. The cells are thus accumulated in masses about the peripheries of these droplets. This substance acts to produce ’ the cancerous organization by thus drawing the scattered cells of the organism into dense stagnant masses. These drops act only until they become saturated with the ergusia. If too great an amount of ergusia is taken away from the individual cells they degenerate. If this degeneration fails and sufficient number of cells are accumulated they can grow in this stagnant area in that they can form and retain suffieient archusia for growth. X-ray, radium, bacteria and animal parasites produce the same stagnant organization in that they liberate growth stimulus in the tissue. Any cells affected by this stimulus grow to form a dense mass of cells free from blood vessels. NATURE OF THE GROWTH STIMULUS IN CANCER 245 In other studies (6) it has been shown that such a dense mass of independently growing cells once established in the organism may continue to reproduce itself through the fact that it can destroy the normal tissues and blood vessels about it. Metas- tases as they occur in cancer can also be explained in the same terms (7). While the cells under ordinary conditions in the body cannot migrate excepting into proteins and fats un- saturated with the ergusia, under conditions of stagnation and crowding such as is seen in cancer conditions are different. The cells in the center of such masses are digested by an excess of the archusia. A soap-like substance is liberated under these conditions which can flow readily over the surface of water solutions, plasma and differentiated cellular tissues in the body. In the films of this soap the cells can migrate readily and grow. In further proof that these mechanical changes in the arrange- ment of cells explain cancer, it is well known that cancerous tissue is a dense mass of stagnant cells. The blood vessels in all cancers are few per unit cell area. They are also constantly being destroyed in this tissue. The author as well as many other students of the tissue culture have shown that all cancerous tissue contains large quantities of the growth stimulus, the archusia (8).

CONDITIONS REUULATING, GROWTH IN THE NORMAL ORGANISM In applying these conditions suitable for growth, differentiation and function in the culture to the body the mechanisms at work in the general development of the organism takes on much simpler aspects. It is in the early periods of development that growth is most active. This growth wanes and ceases and differentiation and function makes its appearance with the development of the blood vascular system. The development of the active circulation not only removes these conditions suitable for growth, but establishes the polarity necessary for function. It became evident then that the dominating forces in the organism which leads to its development and function are associated with the development of the blood vascular system. 246 MONTROSE T. BURROWS There is no evidence that the endothelial cells are possessed with any property of independently growing and forming these structures. The question that presented itself in the further development of cancer was the conditions con trolling and regulating this vascular development. It is the dominance of the crowded masses of cells in the cancer over those forces which maintained the blood vascular system which allowed the cancer to grow and destroy the organism. In studying more carefully the conditions of growth in normal organisms it soon became evident that at no time in the life of these organisms are conditions favorable for an independent growth of the cells as in cancer. At no time is the stagnation and cell density sufficient to allow the cells to grow independently except in the earliest periods of development. At this period the blastomeres are filled with yolk. The simple crowding together of such yolk laden cells in the culture is not ample for their growth. To make them grow the yolk must be removed or a considerable amount of archusia extracted from an actively growing tissue must be added to the medium about them. At a later period after the yolk has disappeared these cells respond by growth when removed from their circulation in the body to the stagnant culture medium. In the embryonic body on the other hand the circulation is too great for them to form and retain ample archusia for their growth. In later life dense masses of cells fail to respond when placed in the plasma clot. Lambert (8) has shown, however, that if fragments of cellular adult tissues are transplanted repeatedly into drops of fresh plasma that they will eventually grow actively in the plasmatic medium. These same tissues can also be made to grow in the first plasma clot by adding an extract of an actively growing tissue or archusia to the medium about them. The question arose then is the growth of the body dependent on conditions different from those found suitable for growth in the cultures and in cancer, or is it controlled by a supply of stimulus or archusia from without. In previous work (9) the author had shown that archusia introduced into an area of the skin of a rat whose circulation has been disturbed, causes the NATURE OF THE GROWTH STIMULUS IN CANCER 247 cells to grow actively and form a dense mass of cells largely free from blood vessels or a cancerous organization. It became evident, therefore, that this same stimulus acting on the egg cell may force channels to develop into the mass of forming blastomeres. These channels may become the blood vascular system (10). Such a stimulus continuing to enter by way of the blood vessels must be in greatest concentration about the endothelial cells of these vessels. This must cause these cells to grow in excess of the cells without. A vascular functioning tissue must, therefore, result. The further proof of this de- duction lay in showing that such a stimulus exists in the universe and acts to so build these metazoan forms. In other studies it has been shown that the storage of fat in the cells, the building of extracellular fibrils, migration and function in cells is the result of their liberating a lipoid-like substance, the ergusia. In studying these various acts in the cultures it became evident that the cells cannot form the ergusia under the conditions suitable for the building of extracellular fibrils by connective tissue, for the absorption of fat, function and migration. Only actively growing cells can maintain their activity indefinitely (11). Connective tissue cells forming extracellular fibrils in the cultures, migrating and taking in fat and heart muscle cells contracting become exhausted of this substance in a few hours or two or three days. In the body this is not true. The heart continues activity for years. The connective tissue cells build large masses of bone and intercellular material over many years in the organism. It was wondered, therefore, whether the ergusia might not also be supplied to these cells from without . It is now known that higher animal life cannot exist on protein, fat, carbohydrates, salts and water alone. Other accessory food substances are also necessary. These have been called vitamins. The most important of these vitamins are A and B. In comparing the properties of the archusia and ergusia with these vitamins, it became evident that they correspond exactly to vitamins B and A respectively. Previous authors had already noted that vitamin A inhibits 248 MONTROSE T. BURROWS the growth of cells. In previous studies the author had noted that the plasma clot induces migration in cells in that it dis- solves the ergusia. It was possible that the failure for the blastomeres to grow when crowded in the cultures was due to the ergusia or vitamin A dissolved in the fat and proteins of the yolk. In previous papers the author has given direct proof that the fat droplets and proteins of the yolk are taken into the cell only as they dissolve the ergusia of the cell. The failure for response of older tissues until after they have been replanted several times into fresh plasma may be due again to the presence of the ergusia or vitamin A in the cells, or intercellular substances of the organism at this period. In proof for this contention it is known that coal tar produces cancer. The author was unable to induce growth in adult tissues by washing them in salt solution. Growth is induced by washing them with or exposing them to fats or ether, but not by substances incapable of dissolving the ergusia. According to these general deductions the ergusia does not inhibit growth as a foreign toxic substance, but as any product of any incomplete reaction may inhibit this reaction when added in excess to the reacting system. The ergusia is a product of the growth reaction, while the archusia is merely the energy necessary for this reaction to take place. Vitamins A and B in the light of these facts are merely products of other growing systems in nature. In proof of these deductions it has become of interest to test solutions rich in archusia for their vitamin activity, to see if the ergusia removed from the body can be replaced by vitamin A, to ascertain directly if the vitamin A content of the normal organism goes up and down with the ability of the cells to react in the cultures, to test for the vitamin content of malignant tumors and to determine directly if vitamin A is a product of inorganic nature or of growing systems. It has been these later studies which have given a definition for vitamins, cancer and a clue to the conditions which have lead to the development of metazoan life in the universe. Careful studies of the archusia content of chick embryos have NATURE OF THE GROWTH STIMULUS IN CANCER 249 shown it highest in 5-day-old chick embryos. It decreasea with the age of the embryos. In the same manner it has been found that the vitamin B content of these embryos is greatest at 5 days and decreases in the same proportion as the archusia decreases (10). The five-day-old embryonic tissue responds at once when placed in the cultures. As tissues age they demand more and more washing with plasma for them to grow. When these embryos are fed to animals the younger are found to contain little or no vitamin A. This A value increases in these embryos with age and their ability to respond in the cultures. The blastomeres again contain very large quantities of vitamin A. This disappears with the yolk granules. Cancerous tissues on the other hand contain no vitamin A but very large quantities of vitamin B (10). In all of these studies we found that the vitamin I3 values vary exactly as the archusia (S) values. In further proof that these substances are identical, Wright (12) has recently shown that the archusia is dialysable as vitamin B is dialysable. Wright (13) has also shown that the yolk of hen’s eggs contains large quantities of this growth stimulus. The quantity in the yolk of a 7-day-old chick embryo is much greater than in the embryo itself. In the studies of the action of coal tar on the organism Jorstad (14) has noticed that rats are readily killed by an over- dose of this substance. He has also found that this toxicity can be overcome by feeding the animals larger quantities of vitamin A. In the studies of the bacterial cultures it has been found that during their active growth they liberate only vitamin B (lo), (15). When they are allowed to overgrow the medium as they do in stagnant pools and in decaying animal and vegetable matter, they suffer a self-digestion as the cells degenerate in cancer. With the breaking up of their protoplasm vitamin A is liberated by them. It is liberated when the protoplasm of these cells break down (11). There is no evidence, therefore, that vitamin A is a product of sun light. It is a product of 250 MONTROSE T. BURROWS living things degenerating in an excess of archusia or vitamin B. Light acts, as shown from the study of X-rays (16), to increase the production of vitamin B in living things. This acts in turn to liberate vitamin A in the tissues (11). The final analysis of the conditions leading to cancer reduces it, therefore, to very definite and practical terms. Cancerous tissue is a simple-growing cellular system. It is identical to a growing culture of bacteria in that it contains no vitamin A and a high value of vitamin B. The normal organism differs from these systems in that it contains a high value of vitamin A. This value decreases in certain periods of embryonic life. The cells at this period resemble the cancer cells in many ways. It was this resemblance which led to the mistaken notion that cancer cells may be growing embryonal cells. In the light of these facts it has become evident that metazoan forms may owe their existence in the universe to the presence of vitamins A and B. Cancer is only the result of a local vitamin imbalance in the organism (17). It may be produced by anything which removes the vitamin A or increases the vitamin B content in the tissues. In the presence of a high B the cell grows and the free A becomes incorporated in the protoplasm, since it is only a product of protoplasmic matter. Coal tar acts to produce cancer in that it removes the vitamin A from the tissues and crowds the cells together in a non- vascular zone so they can produce and retain an excess of archusia (8)or vitamin B. X-rays, radium, anirnal parasites, bacteria, produce cancer in that they increase the vitamin B and disturb the vascular supply to a part so that the vitamin A cannot be supplied to it. The normal actively diffusible stimulus in the organism can then act to stimulate the cells in these parts to grow and form a cancerous organization. Previous authors have given ample evidence to show that metazoan life has come from unicellular forms. How this change was brought about remained obscure. The above observations indicate that it may be due to two things, to the development of a cell capable of absorbing vitamin A and the appearance of this vitamin in the universe. Cancer is a disease which NATURE OF THE GROWTH STIMULUS IN CANCER 251 must be prevalent in an undernourished race and one which suffers from substances and conditions capable of removing vitamin A from their tissues. It must disappear when the nutrition of this race is improved, they cease to be slaves of fashion, have protected themselves against improper drugs, abuses of certain trades and freed themselves from diseases, such as syphilis, which cause undernutrition.

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